1. Field of the Invention
This invention relates to a method for the determination of the concentration of ligands contained in a sample to be analyzed, whereby on at least one support, receptors are immobilized that can enter into a specific bond with the ligands, whereby the sample is brought into contact with the receptors, whereby at least one measurement value representing the frequency of the bonds between the ligands and the receptors is determined and the concentration of the ligands in the sample is determined on the basis of this measurement. The invention also relates to a device for the determination of the concentration of ligands in a sample to be analyzed, with a support, on the surface of which receptors are immobilized that, when they come into contact with the ligands, enter into a specific bond with them, and with at least one detector for the determination of at least one measurement that represents the frequency of the ligand-receptor bonds.
2. Description of the Prior Art
A method of this type and a device of this type are known from Chris A. Rowe-Taitt et al., Biosensors & Bioelectronics 15 (2000), pages 579-589. The device has, as the support, a CCD sensor on whose sensor cells antibodies are immobilized. The antibodies act as receptors which, when they come in contact with the ligands contained in the sample, bond to said ligands, as a result of which the ligand is immobilized on the CCD sensor in the form of a receptor-ligand complex. The antibodies are selected so that they are specific for the ligands, i.e. other biomolecules contained in the sample do not enter into bonds with the receptors when they come into contact with them. A biomolecule can comprise nucleic acids or derivatives thereof (DNA, RNA, PNA, LNA, oligonucleotides, plasmids, chromosomes), peptides, proteins (enzymes, proteins, oligopeptides, cellular receptor proteins and their complexes, peptide hormones, antibodies and fragments thereof, carbohydrates and their derivatives, in particular glycolized proteins and glycosides, fats, fatty acids and/or lipids. After the sample has been brought into contact with the receptors and the ligand has bonded to the receptors, a fluid is applied to the CCD sensor which contains antibodies marked with a fluorescent agent. The antibodies bond to the ligands, as a result of which the ligands are marked with the fluorescent agent. In a further step, the sensor surface with the receptor-ligand complexes immobilized on it is washed to remove the antibodies marked with the fluorescent agent that are not bonded to a receptor from the sensor surface. Then the fluorescent agent is irradiated with optical excitation radiation to excite the emission of fluorescent radiation. The fluorescent radiation thereby released is measured by means of the optical detectors of the CCD sensor. It represents a measurement of the frequency of the ligand-receptor bonds and thus of the concentration of the ligands in the sample.
To make possible a quantitative determination of the concentration of the ligands in the sample, the device must be calibrated. In a calibration method known from actual practice, for this purpose first a dilution series is produced that has a number of calibration samples that contain the ligands in different known concentrations. This can be done, for example, by weighing the ligands, then mixing them with a known quantity of fluid and producing different dilutions from the mixture. To take the calibration measurement, a number of the above mentioned devices for the determination of the concentration of the ligands corresponding to the number of the calibration samples is prepared, whereby it is assumed that these devices are identical to one another and identical to the device used for the actual concentration measurement. By means of these devices, the calibration samples and analyzed, whereby for each of the individual calibration samples, one measurement is taken for the intensity of the fluorescent radiation. In this manner, for each calibration sample a value pair is obtained, consisting of the known concentration of the ligand in the calibration sample and the intensity of the fluorescent radiation measured for this concentration. By means of this value pair, a calibration curve is prepared for which the value pairs form interpolation nodes.
The calibration has the disadvantage that a number of devices that equals the number of calibration samples is required, and these devices can then no longer be used for the measurement of the sample to be analyzed. The calibration of the device is therefore complex and expensive. Another disadvantage of this known method is that for the calibration curve, there are measurements only at the interpolation nodes, and that the profile of the calibration curve between the interpolation nodes must be estimated by interpolation, for example. Finally, however, it is also a problem that the receptors can be unstable, as a result of which the concentration of the receptors that are immobilized on the carrier varies over time. The calibration measurements and the measurement on the sample to be analyzed must therefore be performed as simultaneously as possible, which further complicates the method.
DE 197 36 641 A1 also describes a method of the type described above, in which first a blind measurement is made in which receptors that are immobilized on a silanized glass support are brought into contact with a peroxidase tracer without analyte. From the blind measurement, a measurement for the number of bonding events is determined. This number is significantly lower than the number of receptors that are immobilized on the support, because the peroxidase tracer has a higher molecular weight than the receptors. In addition, calibration solutions of various concentrations are produced that cover the entire measurement range. With these calibration solutions, calibration measurements are measured from which calibration curves are prepared with the value measured in the blind measurement. then the device is regenerated by bringing the receptors that are immobilized on the support with an acid solution, which breaks the bonds between the receptors and the peroxidase tracer. Then the receptors are brought into contact with the sample and measurement values are determined for the frequency of the bonds between the ligands and the receptors. By means of these measurement values and the calibration curves, the concentration of the ligands in the sample is determined. The method has the disadvantage that the receptors can be damaged by contact with the acid solution, as a result of which measurement errors can occur. It is also disadvantageous that the method is relatively time-consuming, because during the calibration, the user has to wait until the bonds between the receptors and the peroxidase tracer have been formed, and the device also has to be regenerated.
The object of the invention is therefore to create a method and a device of the type described above which make it possible to easily determine the concentration of a ligand contained in a sample to be analyzed.